TNF and lymphotoxin beta cooperate in the maintenance of secondary lymphoid tissue microarchitecture but not in the development of lymph nodes

Inactivation of genes encoding members of TNF and TNF receptor families reveal their divergent roles in the formation and function of secondary lymphoid organs. Most lymphotoxin alpha (ltalpha)- and all lymphotoxin beta receptor (ltbetar)-deficient mice are completely devoid of lymph nodes (LNs); however, most lymphotoxin beta (ltbeta)-deficient mice develop mesenteric LNs. Tnf- and tnfrp55-deficient mice develop a complete set of LNs, while ltbeta/tnfrp55 double-deficient mice lack all LNs, demonstrating cooperation between LTbeta and TNFRp55 in LN development. Now we report that ltbeta/tnf double-deficient mice develop the same set of mucosal LNs as do ltbeta-deficient mice, suggesting that ligands other than TNF signal through TNFRp55 during LN development. These LNs retain distinct T and B cells areas; however, they lack follicular dendritic cell networks. Structures resembling germinal centers can be found in the LNs from immunized ltbeta-deficient mice but not in ltbeta/tnf double-deficient mice. Additionally, stromal components of the spleen and LNs appear to be more severely disturbed in ltbeta/tnf double-deficient mice as compared with ltbeta-deficient mice. We conclude that LTbeta and TNF cooperate in the establishment of the correct microarchitecture of lymphoid organs.     

Examination of ras oncoproteins in human plasma from healthy controls and workers exposed to petroleum emissions, including benzene-related compounds

Ras oncoproteins in blood plasma from workers exposed to petroleum emissions and unexposed controls were examined from Polish and Estonian samples. Twenty-four workers and 35 unexposed controls were examined from Poland and 97 exposed and 40 unexposed controls from Estonia. Of the Estonian workers, 50 were exposed to benzene in a benzene production plant and 47 to polyaromatic hydrocarbons and benzene in a cokery. Blood plasma proteins were separated by gel electrophoresis, transferred to a nitrocellulose membrane by Western blotting and detected by chemiluminescence using a monoclonal antibody as the primary antibody. There were no statistically significant differences between the exposed and the control groups in either the Polish or the Estonian samples.     

Multiple mutations in a shuttle vector modified by ultraviolet irradiation, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, and aflatoxin B(1) have different properties than single mutations and may be generated during translesion synthesis

Shuttle vector-based systems are extensively employed to study the mutational properties of various mutagens in mammalian cells. Such vectors are designed for the detection of point mutations, that is small deletions and single base and tandem substitutions. However, mutant target genes carrying two or more point mutations, referred to as multiple mutations, can also be found in various proportions depending on the mutagen and the cells used. To evaluate the frequency and characteristics of multiple mutations, we used a system where the plasmid, pYZ289, was treated by ultraviolet irradiation, aflatoxin B(1) or (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene before transfection into mouse fibroblast cells. The kinds of mutations and the mutational spectra were different for single and multiple mutations. In addition, in at least 75% of the cases, mutations of multiples appeared to arise in the same strand. Furthermore, mutational spectra for multiple mutations were different for 5' and 3' members of multiple sets. These observations suggest that multiple mutations arise via a different mechanism than single mutations. Moreover, these findings suggest that multiples arise during translesion DNA synthesis and involve an error-prone polymerase able to introduce a base opposite misinstructive or noninstructional DNA lesions and subject to subsequent misincorporation errors.     

人蛔虫和猪蛔虫同域种群DNA多态性研究

在我国蛔虫感染遍布各地,受染人数达5亿多.而在多数农村流行区,生猪饲养十分普遍,人群和猪群的蛔虫感染往往并存,且人、猪接触密切.据作者在江西省新建县蔓湖村进行的一年纵向调查,当地人群蛔虫感染率和猪群蛔虫感染率比较接近(约60%-70%),土壤蛔虫卵污染普遍而严重,并长年稳定 ( Peng, W.D. et al. 1996 Ascaris, people and pigs in rural Jiangxi, China. Parasitology 113:547～557.). 因此这些地区人蛔虫和猪蛔虫交叉感染的机会是大量存在的.     

Autoimmune lymphoproliferative syndrome with defective Fas: genotype influences penetrance

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance. Most patients have a heterozygous mutation in the APT1 gene, which encodes Fas (CD95, APO-1), mediator of an apoptotic pathway crucial to lymphocyte homeostasis. Of 17 unique APT1 mutations in unrelated ALPS probands, 12 (71%) occurred in exons 7-9, which encode the intracellular portion of Fas. In vitro, activated lymphocytes from all 17 patients showed apoptotic defects when exposed to an anti-Fas agonist monoclonal antibody. Similar defects were found in a Fas-negative cell line transfected with cDNAs bearing each of the mutations. In cotransfection experiments, Fas constructs with either intra- or extracellular mutations caused dominant inhibition of apoptosis mediated by wild-type Fas. Two missense Fas variants, not restricted to patients with ALPS, were identified. Variant A(-1)T at the Fas signal-sequence cleavage site, which mediates apoptosis less well than wild-type Fas and is partially inhibitory, was present in 13% of African American alleles. Among the ALPS-associated Fas mutants, dominant inhibition of apoptosis was much more pronounced in mutants affecting the intracellular, versus extracellular, portion of the Fas receptor. Mutations causing disruption of the intracellular Fas death domain also showed a higher penetrance of ALPS phenotype features in mutation-bearing relatives. Significant ALPS-related morbidity occurred in 44% of relatives with intracellular mutations, versus 0% of relatives with extracellular mutations. Thus, the location of mutations within APT1 strongly influences the development and the severity of ALPS.     

Identification of caveolin-1 as a fatty acid binding protein

In an attempt to identify high affinity, fatty acid binding proteins present in 3T3-L1 adipocytes plasma membranes, we labeled proteins in purified plasma membranes with the photoreactive fatty acid analogue, 11-m-diazirinophenoxy[11-3H]undecanoate. A single membrane protein of 22 kDa was covalently labeled after photolysis. This protein fractionated with caveolin-1 containing caveolae and was immunoprecipitated by an anti-caveolin-1 monoclonal antibody. Furthermore, 2D-PAGE analysis revealed that both the alpha and beta isoforms of caveolin-1 could be labeled by the photoreactive fatty acid upon photolysis, indicating that both bind fatty acids. The saturable binding of the photoreactive fatty acid suggests caveolin-1 has a lipid binding site that may either operate during intracellular lipid traffic or regulate caveolin-1 function.     

The flattened face of type II beta phosphatidylinositol phosphate kinase binds acidic phospholipid membranes

Type II beta phosphatidylinositol phosphate kinase is a representative phosphatidylinositol phosphate kinase that is active against membrane-bound substrates. The structure of the enzyme contains a flattened basic face that spans the crystallographic dimer interface and is adjacent to the active site. Analytical ultracentrifugation shows that phosphatidylinositol phosphate kinase is a dimer in solution. Modeling suggested that the flattened face binds to acidic phospholipids by electrostatic interactions. The enzyme binds to acidic vesicles containing phosphatidylserine, phosphatidic acid, or phosphoinositides mixed with phosphatidylcholine, but not to neutral phosphatidylcholine vesicles. Binding to acidic vesicles is abolished in the presence of 1.0 M NaCl, consistent with an essential electrostatic contribution to the free energy of binding. The +14 charge on the flattened face of the dimer was reduced to +2 in the triple mutant Lys72Glu/Lys76Glu/Lys78Glu. The mutation has no effect on dimerization, but reduces the apparent KA for 25% phosphatidylserine/75% phosphatidylcholine mixed vesicles by 16-fold. The reduction in the level of binding can be ascribed to a loss of electrostatic interactions based on the finite difference solution to the Poisson-Boltzmann equation. The mutant reduces catalytic activity toward phosphatidylinositol 5-phosphate by approximately 50-fold. The wild-type enzyme binds half-maximally to phosphatidylinositol 4,5-bisphosphate-containing vesicles at a mole fraction of 0.3% in a phosphatidylcholine background, as compared to a 22% mole fraction in phosphatidylserine. The binding to phosphatidylinositol 4,5-bisphosphate-containing membranes is less sensitive to salt and to the triple mutation than binding to phosphatidylserine-containing membranes, suggesting that at least part of phosphatidylinositol 4,5-bisphosphate's interaction with the enzyme is independent of the flattened face. It is concluded that the flattened face of type II beta phosphatidylinositol phosphate kinase binds to membranes through nonspecific interactions, and that this interaction is essential for efficient catalysis.     

Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells

Multipotent and self-renewing neural stem cells have been isolated in culture, but equivalent cells have not yet been prospectively identified in neural tissue. Using cell surface markers and flow cytometry, we have isolated neural crest stem cells (NCSCs) from mammalian fetal peripheral nerve. These cells are phenotypically and functionally indistinguishable from NCSCs previously isolated by culturing embryonic neural tube explants. Moreover, in vivo BrdU labeling indicates that these stem cells self-renew in vivo. NCSCs freshly isolated from nerve tissue can be directly transplanted in vivo, where they generate both neurons and glia. These data indicate that neural stem cells persist in peripheral nerve into late gestation by undergoing self-renewal. Such persistence may explain the origins of some PNS tumors in humans.     

Pulsed-light inactivation of food-related microorganisms

The effects of high-intensity pulsed-light emissions of high or low UV content on the survival of predetermined populations of Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus were investigated. Bacterial cultures were seeded separately on the surface of tryptone soya-yeast extract agar and were reduced by up to 2 or 6 log10 orders with 200 light pulses (pulse duration, approximately 100 ns) of low or high UV content, respectively (P < 0.001).     

Contact analysis of a posterior cervical spine plate using a three-dimensional canine finite element model

The development of a three-dimensional finite element model of a posteriorly plated canine cervical spine (C3-C6) including contact nonlinearities is described. The model was created from axial CT scans and the material properties were derived from the literature. The model demonstrated sufficient accuracy from the results of a mesh convergence test. Significant steps were taken toward establishing model validation by comparison of plate surface strains with a posteriorly plated canine cervical spine under three-point bending. This model was developed to better characterize the contact pressures at the various interfaces under average physiologic canine loading. The analysis showed that the screw-plate interfaces had the highest values of all the mechanical parameters evaluated.